Evolution of DNA Sequencing by Jonathan Eisen

Evolution of Sequencing
Workshop in Applied Phylogenetics
March 8, 2015
Bodega Bay Marine Lab
Jonathan A. Eisen
UC Davis Genome Center
1

Notes
• Some slides have been kept in here for
historical information - do not
necessarily use them all in
presentations
• For most of the methods presented, the
source material can be genomic DNA,
PCR amplified DNA, RNA converted into
cDNA, etc.
• I have attempted to include the source
of all materials - apologies when this is
not done or done incorrectly
15

Review Papers
16
Mardis ER. Next-generation sequencing platforms.
Annu Rev Anal Chem 2013;6:287-303.  
doi: 10.1146/annurev-anchem-062012-092628.
Next-Generation DNA
Sequencing Methods
Elaine R. Mardis
Departments of Genetics and Molecular Microbiology and Genome Sequencing Center,
Washington University School of Medicine, St. Louis MO 63108; email: emardis@wustl.edu
Annu. Rev. Genomics Hum. Genet. 2008.
9:387–402
First published online as a Review in Advance on
June 24, 2008
The Annual Review of Genomics and Human Genetics
is online at genom.annualreviews.org
This article’s doi:
10.1146/annurev.genom.9.081307.164359
Copyright c⃝ 2008 by Annual Reviews.
All rights reserved
1527-8204/08/0922-0387$20.00
Key Words
massively parallel sequencing, sequencing-by-synthesis, resequencing
Abstract
Recent scientiﬁc discoveries that resulted from the application of next-
generation DNA sequencing technologies highlight the striking impact
of these massively parallel platforms on genetics. These new meth-
ods have expanded previously focused readouts from a variety of DNA
preparation protocols to a genome-wide scale and have ﬁne-tuned their
resolution to single base precision. The sequencing of RNA also has
transitioned and now includes full-length cDNA analyses, serial analysis
of gene expression (SAGE)-based methods, and noncoding RNA dis-
Click here for quick links to
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including:
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FurtherANNUAL
REVIEWS
Annu.Rev.Genom.HumanGenet.2008.9:387-402.Downloadedfromarjournals.annualreviews.org
byUniversidadNacionalAutonomadeMexicoon11/17/09.Forpersonaluseonly.
Annu. Rev. Genomics Hum. Genet. 2008.
9:387–402
First published online as a Review in Advance on
June 24, 2008
The Annual Review of Genomics and Human Geneti
is online at genom.annualreviews.org
This article’s doi:
10.1146/annurev.genom.9.081307.164359
Copyright c⃝ 2008 by Annual Reviews.
All rights reserved
1527-8204/08/0922-0387$20.00
Annu.Rev.Genom.HumanGenet.2008.9:3
byUniversidadNacionalAutonoma• Open Access Review Papers
• http://www.microbialinformaticsj.com/content/2/1/3/
• http://www.hindawi.com/journals/bmri/2012/251364/abs/
• http://m.cancerpreventionresearch.aacrjournals.org/content/
5/7/887.full

17
Approaching to NGS
Discovery of DNA structure
(Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31)
1953
Sanger sequencing method by F. Sanger
(PNAS ,1977, 74: 560-564)
1977
PCR by K. Mullis
(Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73)
1983
Development of pyrosequencing
(Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365)
1993
1980
1990
2000
2010
Single molecule emulsion PCR 1998
Human Genome Project
(Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351)
Founded 454 Life Science 2000
454 GS20 sequencer
(First NGS sequencer)
2005
Founded Solexa 1998
Solexa Genome Analyzer
(First short-read NGS sequencer)
2006
GS FLX sequencer
(NGS with 400-500 bp read lenght)
2008
Hi-Seq2000
(200Gbp per Flow Cell)
2010
Illumina acquires Solexa
(Illumina enters the NGS business)
2006
ABI SOLiD
(Short-read sequencer based upon ligation)
2007
Roche acquires 454 Life Sciences
(Roche enters the NGS business)
2007
NGS Human Genome sequencing
(First Human Genome sequencing based upon NGS technology)
2008
Miseq
Roche Jr
Ion Torrent
PacBio
Oxford
From Slideshare presentation of Cosentino Cristian
http://www.slideshare.net/cosentia/high-throughput-equencing
Sequencing Technology Timeline

18
Gen 0:
Proto
Sequencing

20
Gen 0:
Proto
Sequencing

Gen 1: Manual Sequencing
21
Gen 0:
Proto
Sequencing

21
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
Gen 1:
Manual
Sequencing

Maxam-Gilbert Sequencing
22
From http://www.pnas.org/content/74/2/560.full.pdf

Sanger Sequencing of PhiX174
23
http://www.ncbi.nlm.nih.gov/
pmc/articles/PMC431765/

Nobel Prize 1980: Berg, Gilbert, Sanger
26
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1980/

27
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
Gen 1:
Manual
Sequencing

Gen 2: Automated Sanger
28
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
Gen 1:
Manual
Sequencing

Gen 2: Automated Sanger
28
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger

Automation of Sanger Part I
29
Sanger method with labeled dNTPs
The Sanger mehtods is based on the idea that inhibitors can
terminate elongation of DNA at specific points

Many Systems for Sanger Automation
30
Thanks to Robin Coope and Dale Yazuki for comments on 2014 talk
ABI 3700 ABI 3730 ABI 3730xl
Megabase 1000 Megabase 4000 LiCor

Automation of Sanger Part II
31

Some Automated Sanger Highlights
• 1991: ESTs by Venter
• 1995: H. influenzae shotgun genome
• 1996: Yeast, archaeal genomes
• 1998: 1st animal genome - C. elegans
• 1999: Drosophila shotgun genome
• 2000: Arabidopsis genome
• 2000: Human genome
• 2004: Shotgun metagenomics
32
Thanks to Keith Bradnam for C. elegans suggestion

Gen 3: Clusters not Clones (NextGen)
33
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger

33
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
Clones

33
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
454-
Roche
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
Clones

33
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
Solexa-
Illumina
454-
Roche
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
Clones

33
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
ABI-
Solid
Solexa-
Illumina
454-
Roche
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
Clones

33
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
ABI-
Solid
Solexa-
Illumina
454-
Roche
Ion
Torrent
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
Clones

Generation III: Clusters not Clones
34

Generation III = “NextGen”
35

NextGen Sequencing Outline
36
Next-generation sequencing platforms
Isolation and purification of
target DNA
Sample preparation
Library validation
Cluster generation
on solid-phase
Emulsion PCR
Sequencing by synthesis
with 3’-blocked reversible
terminators
Pyrosequencing Sequencing by ligation
Single colour imaging
Sequencing by synthesis
with 3’-unblocked reversible
terminators
AmplificationSequencingImaging
Four colour imaging
Data analysis
Roche 454Illumina GAII ABi SOLiD Helicos HeliScope

37
Pyrosequencing
Sanger
method
-
ABi SOLiD
HeliScope
Nanopore
Roche 454
lumina GAII
From Slideshare presentation of
Cosentino Cristian
http://www.slideshare.net/cosentia/high-
throughput-equencing
NextGen #1: 454

38
Pyrosequencing
Sanger
method
-
ABi SOLiD
HeliScope
Nanopore
Roche 454
lumina GAII
Cosentino Cristian
NextGen #1: Roche 454

39
Pyrosequencing
Sanger
method
-
ABi SOLiD
HeliScope
Nanopore
Roche 454
lumina GAII
Cosentino Cristian
NextGen #1: Roche 454

454 -> Roche
• 1st “Next-generation” sequencing
system to become commercially
available, in 2004
• Uses pyrosequencing
• Polymerase incorporates nucleotide
• Pyrophosphate released
• Eventually light from luciferase released
• Three main steps in 454 method
• Library prep
• Emulsion PCR
• Sequencing
40

41
Roche 454 Wokflow
From http://acb.qfab.org/acb/ws09/presentations/Day1_DMiller.pdf
http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications

a
b
DNA library preparation
Emulsion PCR
A
A
A B
B
B
4.5 hours
8 hours
Ligation
Selection
(isolate AB
fragments
only)
•Genome fragmented
by nebulization
•No cloning; no colony
picking
•sstDNA library created
with adaptors
•A/B fragments selected
using avidin-biotin
purification
gDNA sstDNA library
gDNA
fragmented by
nebulization
or sonication
Fragments are end-
repaired and ligated to
adaptors containing
universal priming sites
Fragments are denatured and
AB ssDNA are selected by
avidin/biotin purification
(ssDNA library)
From Mardis 2008. Annual Rev. Genetics 9: 387.
Roche 454 Step 1: Libraries
42

Anneal sstDNA to an excess of
DNA capture beads
Emulsify beads and PCR
reagents in water-in-oil
microreactors
Clonal amplification occurs
inside microreactors
Break microreactors and
enrich for DNA-positive
beads
b
c
Emulsion PCR
Sequencing
A B
8 hours
7.5 hours
using avidin-biotin
purification
gDNA sstDNA library
sstDNA library Bead-amplified sstDNA library
Roche 454 Step 2: Emulsion PCR
43

DNA capture beads reagents in water-in-oil
microreactors
inside microreactors enrich for DNA-positive
beads
Amplified sstDNA library beads Quality filtered bases
c
Sequencing
7.5 hours
sstDNA library Bead-amplified sstDNA library
•Well diameter: average of 44 µm
•400,000 reads obtained in parallel
•A single cloned amplified sstDNA
bead is deposited per well
390 Mardis
Roche 454 Step 3: Pyrosequencing
44

Pyrosequencing
44 µm
Pyrosequecning
Reads are recorded as flowgrams
Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402
Nature Reviews genetics, 2010, 11: 31-46
Sanger
method
-
ABi SOLiD
HeliScope
Nanopore
Roche 454
Illumina GAII
Roche 454 Step 3: Pyrosequencing
45

Roche 454 Key Issues
• Number of repeated nucleotides
estimated by amount of light ... many
errors
• Reasonable number of failures in EM-
PCR and other steps
46

Roche 454 Evolution
47

NextGen #2: Solexa
48
Sequecning by synthesis with reversible terminator
anger
ethod
he 454
SOLiD
iScope
nopore
-

NextGen #2: Solexa Illumina
Sequecning by synthesis with reversible terminator
anger
ethod
he 454
SOLiD
iScope
nopore
-
49

NextGen #2: Illumina Accessories
50
Cluster station
Genome Analyzer IIxPaired-end module Linux server
Bioanalyzer 2100
Instrumentation
mple
aration
sters
fication
ncing by
thesis
alysis
eline
duction
na GAII
igh
ughput

Illumina Outline
51
Clusters
amplification
Clusterstation
Wash cluster
station
Clustergeneration
Linearization,
Blocking and
primer
Hybridization
Read 1
Prepare read 2
Read 2
GAIIx&PE
SBSsequencing
Pipeline base call
Data analysis
Sample
preparation and
library validation
Analysis
Sequencing workflow
Sample
preparation
Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
Introduction
Illumina GAII
High
throughput
From Slideshare
presentation of
Cosentino Cristian
http://
www.slideshare.net/
cosentia/high-

Illumina Step 1: Prep & Attach DNA
52
termined by user-deﬁned instrument settings,
which permits discrete read lengths of 25–35
the Illumina data from each run, removing
poor-quality sequences.
Adapter
DNA fragment
Dense lawn
of primers
Adapter
Attached
DNA
Adapters
Prepare genomic DNA sample
Randomly fragment genomic DNA
and ligate adapters to both ends of
the fragments.
Attach DNA to surface
Bind single-stranded fragments
randomly to the inside surface
of the flow cell channels.
Nucleotides
a
Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a
nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp
are isolated via gel extraction and amplified using limited cycles of PCR

Illumina Step 2: Clusters by Bridge PCR
53
Attached
Prepare genomic DNA sample
Randomly fragment genomic DNA
and ligate adapters to both ends of
the fragments.
Attach DNA to surface
Bind single-stranded fragments
randomly to the inside surface
of the flow cell channels.
Bridge amplification
Add unlabeled nucleotides
and enzyme to initiate solid-
phase bridge amplification.
Denature the double
stranded molecules
Nucleotides
Figure 2
The Illumina sequencing-by-synthesis approach. Cluster strands created by bridge amplification are primed and all four fluorescently
labeled, 3′-OH blocked nucleotides are added to the flow cell with DNA polymerase. The cluster strands are extended by one
nucleotide. Following the incorporation step, the unused nucleotides and DNA polymerase molecules are washed away, a scan buffer is
added to the flow cell, and the optics system scans each lane of the flow cell by imaging units called tiles. Once imaging is completed,
chemicals that effect cleavage of the fluorescent labels and the 3′-OH blocking groups are added to the flow cell, which prepares the
cluster strands for another round of fluorescent nucleotide incorporation.
392 Mardis
• From : http://seqanswers.com/forums/showthread.php?t=21. Steps 2-6: Cluster Generation by Bridge Amplification. In contrast to the 454 and ABI
methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on
the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters
ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to
reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the
surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow
cell surface. This process occurs in what is referred to as Illumina's "cluster station", an automated flow cell processor.

55
25–35 bp, and each sequencing run yields be-
tween 2–4 Gb of DNA sequence data. Once
generation read type has a unique error model
different from that already established for
b
Laser
First chemistry cycle:
determine first base
To initiate the first
sequencing cycle, add
all four labeled reversible
terminators, primers, and
DNA polymerase enzyme
to the flow cell.
Image of first chemistry cycle
After laser excitation, capture the image
of emitted fluorescence from each
cluster on the flow cell. Record the
identity of the first base for each cluster.
Sequence read over multiple chemistry cycles
Repeat cycles of sequencing to determine the sequence
Before initiating the
next chemistry cycle
The blocked 3' terminus
and the fluorophore
from each incorporated
base are removed.
GCTGA...
Illumina Step 3: Sequencing by Synthesis
From : http://seqanswers.com/forums/showthread.php?t=21. Steps 7-12: Sequencing by Synthesis. A flow cell containing millions of unique clusters
is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation
of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first
base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four
bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base
extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful
Illumina reads range from 26-50 bases.

56
SBS technology
Sample
eparation
Clusters
mplification
quencing by
synthesis
Analysis
pipeline
troduction
umina GAII
High
roughput
Illumina Step 3: Sequencing by Synthesis

57
Laser
terminators, primers, and
DNA polymerase enzyme
to the flow cell.
Image of first chemistry cycle
After laser excitation, capture the image
of emitted fluorescence from each
cluster on the flow cell. Record the
identity of the first base for each cluster.
Sequence read over multiple chemistry cycles
Repeat cycles of sequencing to determine the sequence
of bases in a given fragment a single base at a time.
Before initiating the
next chemistry cycle
The blocked 3' terminus
and the fluorophore
from each incorporated
base are removed.
GCTGA...
Figure 2
(Continued )
www.annualreviews.org • Next-Generation DNA Sequencing Methods 393
Illumina Step 3: Cycling

Illumina Evolution
58

NextGen #3: 454: ABI Solid
62
Sequecning by ligation
Sanger
method
Roche 454
-
HeliScope
Nanopore
ABi SOLiD
Illumina GAII
From Slideshare
presentation of
Cosentino Cristian
http://
www.slideshare.net/
cosentia/high-

ABI Solid Details
63
3-GG09-20 ARI 25 July 2008 14:57
A C G T
1stbase
2nd base
A
C
G
T
3'TAnnnzzz5'
3'TCnnnzzz5'
3'TGnnnzzz5'
3'TTnnnzzz5'
Cleavage site
Di base probesSOLiD™ substrate
3'
TA
AT
Universal seq primer (n)
3'
P1 adapter Template sequence
POH
Universal seq primer (n–1)
Ligase
Phosphatase
+
1. Prime and ligate
2. Image
4. Cleave off fluor
5. Repeat steps 1–4 to extend sequence
3'
Universal seq primer (n–1)
1. Melt off extended
sequence
2. Primer reset3'
AA AC G
G GG
C C
C
T AA
A GG
CC
T TTT
6. Primer reset
7. Repeat steps 1–5 with new primer
8. Repeat Reset with , n–2, n–3, n–4 primers
TA
AT
AT
3'
TA
AT
3'
Excite Fluorescence
Cleavage agent
P
HO
TA
AA AG AC AAAT
TT TC TG TT AC
TG
CG
GC
3'
3. Cap unextended strands
3'
PO4
1 2 3 4 5 6 7 ... (n cycles)Ligation cycle
3'
3'1 μm
bead
1 μm
bead
1 μm
bead
–1
Universal seq primer (n)
3'
1
Primer round 1
Template
Primer round 2 1 base shift
Glass slide
3'5' Template sequence
1 μm
bead P1 adapter
Read position 35343332313029282726252423222120191817161514131211109876543210
a
The ligase-mediated sequencing approach of the
Applied Biosystems SOLiD sequencer. In a manner
similar to Roche/454 emulsion PCR amplification, DNA
fragments for SOLiD sequencing are amplified on the
surfaces of 1-μm magnetic beads to provide sufficient
signal during the sequencing reactions, and are then
deposited onto a flow cell slide. Ligase-mediated
sequencing begins by annealing a primer to the shared
adapter sequences on each amplified fragment, and
then DNA ligase is provided along with specific
fluorescent- labeled 8mers, whose 4th and 5th bases
are encoded by the attached fluorescent group. Each
ligation step is followed by fluorescence detection, after
which a regeneration step removes bases from the
ligated 8mer (including the fluorescent group) and
concomitantly prepares the extended primer for another
round of ligation. (b) Principles of two-base encoding.
Because each fluorescent group on a ligated 8mer
identifies a two-base combination, the resulting
sequence reads can be screened for base-calling
errors versus true polymorphisms versus single base
deletions by aligning the individual reads to a known
high-quality reference sequence.
From Mardis 2008. Annual Rev.
Genetics 9: 387.

ABI Solid Evolution
64

Complete Genomics
65
REVIEW
ased the number of false positive gene
ely reduced the number gene candidates
sitosterolemia phenotype were determined after comparison of
the patient’s genome to a collection of reference genomes.
Ultimately, it was determined that the patient failed the standard
omplete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DNB
f the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a set
onding to 5 bases from the distinct adapter site. Both standard and extended anchor schemes are shown. Reprinted with
pyright XXXX American Association for the Advancement of Science.
gure 3. Schematic of Complete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DN
nthesis, and dimensions of the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a
common probes corresponding to 5 bases from the distinct adapter site. Both standard and extended anchor schemes are shown. Reprinted w
Figure 3. Schematic of Complete
Genomics’ DNB array generation and
cPAL technology. (A) Design of sequencing
fragments, subsequent DNB synthesis, and
dimensions of the patterned nanoarray
used to localize DNBs illustrate the DNB
array formation. (B) Illustration of
sequencing with a set of common probes
corresponding to 5 bases from the distinct
adapter site. Both standard and extended
anchor schemes are shown.
From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.

Comparison in 2008
66
Roche (454) Illumina SOLiD
Chemistry Pyrosequencing Polymerase-based Ligation-based
Amplification Emulsion PCR Bridge Amp Emulsion PCR
Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb
Mb/run 100 Mb 1300 Mb 3000 Mb
Time/run 7 h 4 days 5 days
Read length 250 bp 32-40 bp 35 bp
Cost per run
(total)
$8439 $8950 $17447
Cost per Mb $84.39 $5.97 $5.81
From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/
CSHL_nextgen.ppt 66

Comparison in 2012
67
Roche (454) Illumina SOLiD
Chemistry Pyrosequencing Polymerase-based Ligation-based
Amplification Emulsion PCR Bridge Amp Emulsion PCR
Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb
Mb/run 100 Mb 1300 Mb 3000 Mb
Time/run 7 h 4 days 5 days
Read length 250 bp 32-40 bp 35 bp
Cost per run
(total)
$8439 $8950 $17447
Cost per Mb $84.39 $5.97 $5.81
From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/
CSHL_nextgen.ppt 67

Applied Biosystems Ion Torrent PGM
70

Applied Biosystems Ion Torrent PGM
71
Workflow similar to
that for Roche/454
systems.
Not surprising,
since invented by
people from 454.

Ion Torrent pH Based Sequencing
72
Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.

Multiplexing
75
From http://www.illumina.com/technology/multiplexing_sequencing_assay.ilmn

Multiplexing
76
http://res.illumina.com/documents/products/datasheets/datasheet_sequencing_multiplex.pdf

Small Amounts of DNA
77
http://www.epibio.com/docs/default-source/protocols/nextera-dna-sample-prep-kit-(illumina--compatible).pdf?sfvrsn=4

Capture Methods
78
High throughput sample preparation
Sample
preparation
Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
Introduction
Illumina GAII
High
throughput
Nature Methods, 2010, 7: 111-118
RainDance
Microdroplet PCR
Roche Nimblegen
Salid-phase capture with custom-
designed oligonucleotide microarray
Reported 84% of
capture efficiency
Reported 65-90% of capture efficiency

79
High throughput sample preparation
Sample
preparation
Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
Introduction
Illumina GAII
High
throughput
Agilent SureSelect
Solution-phase capture with
streptavidin-coated magnetic beads
Reported 60-80% of capture efficiency
Cosentino Cristian
http://www.slideshare.net/cosentia/
high-throughput-equencing
Capture Methods

Illumina Paired Ends
80
Paired-end technology
Paired-end sequencing works into GA and uses chemicals from the PE
module to perform cluster amplification of the reverse strandSample
preparation
Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
Introduction
Illumina GAII
High
throughput
From Slideshare
presentation of Cosentino
Cristian
http://www.slideshare.net/
cosentia/high-throughput-
equencing

Moleculo
81
Large fragments
DNA

Moleculo
81
Large fragments
DNA
Isolate and amplify

Moleculo
81
Large fragments
DNA
Isolate and amplify
Sublibrary w/ unique barcodes

Moleculo
81
Large fragments
DNA
Isolate and amplify
CACC GGAA TCTC ACGT AAGG GATC AAAA

Moleculo
81
Large fragments
DNA
Isolate and amplify
Sequence w/ Illumina

Moleculo
81
Large fragments
DNA
Isolate and amplify
Sequence w/ Illumina
Assemble seqs w/ same codes

HiC
82
Suggested by Carlos Bustamante and Keith Bradnam

HiC Crosslinking & Sequencing
Beitel CW, Froenicke L, Lang JM, Korf IF, Michelmore
RW, Eisen JA, Darling AE. (2014) Strain- and plasmid-
level deconvolution of a synthetic metagenome by
sequencing proximity ligation products. PeerJ 2:e415
http://dx.doi.org/10.7717/peerj.415
Table 1 Species alignment fractions. The number of reads aligning to each replicon present in the
synthetic microbial community are shown before and after filtering, along with the percent of total
constituted by each species. The GC content (“GC”) and restriction site counts (“#R.S.”) of each replicon,
species, and strain are shown. Bur1: B. thailandensis chromosome 1. Bur2: B. thailandensis chromosome
2. Lac0: L. brevis chromosome, Lac1: L. brevis plasmid 1, Lac2: L. brevis plasmid 2, Ped: P. pentosaceus,
K12: E. coli K12 DH10B, BL21: E. coli BL21. An expanded version of this table can be found in Table S2.
Sequence Alignment % of Total Filtered % of aligned Length GC #R.S.
Lac0 10,603,204 26.17% 10,269,562 96.85% 2,291,220 0.462 629
Lac1 145,718 0.36% 145,478 99.84% 13,413 0.386 3
Lac2 691,723 1.71% 665,825 96.26% 35,595 0.385 16
Lac 11,440,645 28.23% 11,080,865 96.86% 2,340,228 0.46 648
Ped 2,084,595 5.14% 2,022,870 97.04% 1,832,387 0.373 863
BL21 12,882,177 31.79% 2,676,458 20.78% 4,558,953 0.508 508
K12 9,693,726 23.92% 1,218,281 12.57% 4,686,137 0.507 568
E. coli 22,575,903 55.71% 3,894,739 17.25% 9,245,090 0.51 1076
Bur1 1,886,054 4.65% 1,797,745 95.32% 2,914,771 0.68 144
Bur2 2,536,569 6.26% 2,464,534 97.16% 3,809,201 0.672 225
Bur 4,422,623 10.91% 4,262,279 96.37% 6,723,972 0.68 369
Figure 1 Hi-C insert distribution. The distribution of genomic distances between Hi-C read pairs is
shown for read pairs mapping to each chromosome. For each read pair the minimum path length on
the circular chromosome was calculated and read pairs separated by less than 1000 bp were discarded.
The 2.5 Mb range was divided into 100 bins of equal size and the number of read pairs in each bin
was recorded for each chromosome. Bin values for each chromosome were normalized to sum to 1 and
plotted.
E. coli K12 genome were distributed in a similar manner as previously reported (Fig. 1;
(Lieberman-Aiden et al., 2009)). We observed a minor depletion of alignments spanning
the linearization point of the E. coli K12 assembly (e.g., near coordinates 0 and 4686137)
due to edge eVects induced by BWA treating the sequence as a linear chromosome rather
than circular.
10.7717/peerj.415 9/19
Figure 2 Metagenomic Hi-C associations. The log-scaled, normalized number of Hi-C read pairs
associating each genomic replicon in the synthetic community is shown as a heat map (see color scale,
blue to yellow: low to high normalized, log scaled association rates). Bur1: B. thailandensis chromosome
1. Bur2: B. thailandensis chromosome 2. Lac0: L. brevis chromosome, Lac1: L. brevis plasmid 1, Lac2:
L. brevis plasmid 2, Ped: P. pentosaceus, K12: E. coli K12 DH10B, BL21: E. coli BL21.
reference assemblies of the members of our synthetic microbial community with the same
alignment parameters as were used in the top ranked clustering (described above). We first
Figure 3 Contigs associated by Hi-C reads. A graph is drawn with nodes depicting contigs and edges
depicting associations between contigs as indicated by aligned Hi-C read pairs, with the count thereof
depicted by the weight of edges. Nodes are colored to reflect the species to which they belong (see legend)
with node size reflecting contig size. Contigs below 5 kb and edges with weights less than 5 were excluded.
Contig associations were normalized for variation in contig size.
typically represent the reads and variant sites as a variant graph wherein variant sites are
represented as nodes, and sequence reads define edges between variant sites observed in
the same read (or read pair). We reasoned that variant graphs constructed from Hi-C
data would have much greater connectivity (where connectivity is defined as the mean
path length between randomly sampled variant positions) than graphs constructed from
mate-pair sequencing data, simply because Hi-C inserts span megabase distances. Such
Figure 4 Hi-C contact maps for replicons of Lactobacillus brevis. Contact maps show the number of
Hi-C read pairs associating each region of the L. brevis genome. The L. brevis chromosome (Lac0, (A),
Chris Beitel
@datscimed
Aaron Darling
@koadman

Dovetail “Chicago” Libraries
84
http://arxiv.org/abs/1502.05331
Suggested by

Single Cell Sequencing
85
Suggested by Keith Robison

Gen 4: Single Molecule
86
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
ABI-
Solid
Solexa-
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454-
Roche
Ion
Torrent
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
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86
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Maxam-Gilbert
ABI-
Solid
Solexa-
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454-
Roche
Ion
Torrent
Gen 1:
Manual
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Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
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Gen 4:
Single
Molecule

Helicos
86
Gen 0:
Proto
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Sanger
Maxam-Gilbert
ABI-
Solid
Solexa-
Illumina
454-
Roche
Ion
Torrent
Gen 1:
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Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
Clones
Gen 4:
Single
Molecule

Pacbio
Helicos
86
Gen 0:
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Maxam-Gilbert
ABI-
Solid
Solexa-
Illumina
454-
Roche
Ion
Torrent
Gen 1:
Manual
Sequencing
Gen 2:
Automated
Sanger
Gen 3:
Clusters
Not
Clones
Gen 4:
Single
Molecule

Oxford
NanoPore
Pacbio
Helicos
86
Gen 0:
Proto
Sequencing
Sanger
Maxam-Gilbert
ABI-
Solid
Solexa-
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454-
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Gen 2:
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Gen 4:
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Molecule

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3rd
Generation Sequencing
y
w for
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ent_sequencing

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Single Molecule II: Pacific Biosciences

89
Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.

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Φ29 polymerase. Each amplified product of a circularized
fragment is called a DNA nanoball (DNB). DNBs are selectively
attached to a hexamethyldisilizane (HMDS) coated silicon chip
that is photolithographically patterned with aminosilane active
sites. Figure 3A illustrates the DNB array design.
The use of the DNBs coupled with the highly patterned array
offers several advantages. The production of DNBs increases
signal intensity by simply increasing the number of hybridization
sites available for probing. Also, the size of the DNB is on the
same length scale as the active site or “sticky” spot patterned on
Each hybridization and ligation cycle is followed by fluorescent
imaging of the DNB spotted chip and subsequently regeneration
of the DNBs with a formamide solution. This cycle is repeated
until the entire combinatorial library of probes and anchors is
examined. This formula of the use of unchained reads and
regeneration of the sequencing fragment reduces reagent con-
sumption and eliminates potential accumulation errors that can
arise in other sequencing technologies that require close to
completion of each sequencing reaction.19,52,53
Complete Genomics showcased their DNB array and cPAL
Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing a single DNA
polymerase (Φ29) bound to the bottom glass surface. The ZMW and the confocal imaging system allow fluorescence detection only at the bottom
surface of each ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation on to a sequencing template. The corresponding
temporal fluorescence detection with respect to each of the five incorporation steps is shown below. Reprinted with permission from ref 39. Copyright
2009 American Association for the Advancement of Science.
Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing
a single DNA polymerase (Φ29) bound to the bottom glass surface. The ZMW and the confocal imaging system allow fluorescence
detection only at the bottom surface of each ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation
on to a sequencing template. The corresponding temporal fluorescence detection with respect to each of the five incorporation steps
is shown below.

Why Finish Genomes?
91
The Value of Finished Bacterial Genomes
Why Are Finished Genomes So Important?
When Sanger sequencing was the only available sequencing technique, it was expensive — but not unusual — to
improve genome drafts until they were good enough to be considered finished. With the availability of short-read
sequencing technologies, draft genomes became cheap and easy to produce, and the majority of researchers
skipped the more labor- and time-intensive task of finishing genomes, with the realization that critical data
may be missing (Figure 3). Finished genomes are crucial for understanding microbes and advancing the field of
microbiology3
because:
• Functional genomic studies demand a high-quality,
complete genome sequence as a starting point
• Comparative genomics is meaningful only in terms
of complete genome sequences
• Understanding genome organization provides
biological insights
• Microbial forensics requires at least one complete
reference genome sequence
• Finished genomes aid in microbial outbreak source
identification and phylogenetic analysis
• A complete genome is a permanent scientific
resource
Figure 3: History of drafted vs. finished genomes (adapted from ref. 2).
Microbial Genetics Using SMRT Sequencing
0
2000
4000
6000
8000
10000
12000
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Numberofgenomes
Drafted Bacterial Genomes
Finished Bacterial Genomes

Why Finish Genomes
92
JOURNAL OF BACTERIOLOGY, Dec. 2002, p. 6403–6405 Vol. 184, No. 23
0021-9193/02/$04.00ϩ0 DOI: 10.1128/JB.184.23.6403–6405.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
DIALOG
The Value of Complete Microbial Genome Sequencing
(You Get What You Pay For)
Claire M. Fraser,* Jonathan A. Eisen, Karen E. Nelson, Ian T. Paulsen,
and Steven L. Salzberg
The Institute for Genomic Research, Rockville, Maryland 20850
Since the publication of the complete Haemophilus influen-
zae genome sequence in July 1995 (4), the field of microbiology
has been one of the largest beneficiaries of the breakthroughs
in genomics and computational biology that made this accom-
plishment possible. When the 1.8-Mbp H. influenzae project
began in 1994, it was not certain that the whole-genome shot-
gun sequencing strategy would succeed because it had never
been attempted on any piece of DNA larger than an average
lambda clone (ϳ40 kbp) (9).
During the past 7 years, progress in DNA sequencing tech-
nology, the design of new vectors for library construction for
use in shotgun sequencing projects, significant improvements
in closure and finishing strategies, and more sophisticated and
robust methods for gene finding and annotation have dramat-
ically reduced the time required for each stage of a genome
project and the cost per base pair while at the same time
organisms to be sampled because of the cost savings that would
come from not taking each project to completion. While this
strategy does achieve a cost savings, today it is only approxi-
mately 50%, and this comes at a cost in terms of the quality and
utility of the finished product.
A complete genome sequence represents a finished product
in which the order and accuracy of every base pair have been
verified. In contrast, a draft sequence, even one of high cov-
erage, represents a collection of contigs of various sizes, with
unknown order and orientation, that contain sequencing errors
and possible misassemblies. As stated by Selkov et al. in a 2000
paper on a draft sequence of Thiobacillus ferrooxidans, “It is
clear that such sequencing. . .produces more errors than com-
plete genome sequencing. . . . The current error rate is esti-
mated to be 1 per 1,000 to 2,000 base pairs vs. 1 in 10,000 base
pairs for complete sequencing” (10). In fact, the difference is
http://jb.asm.org/Downloadedfrom
http://jb.asm.org/content/184/23/6403.full

HGAP Assembly from PacBio
93
PacBio assembly CDC assembly
Illumina assemblySanger validation
HGAP Assembler for PacBio Data
http://www.pacificbiosciences.com/pdf/
microbial_primer.pdf

Detecting Modified Bases
94
Page 2 www.pacb.com/basemod
processes.
The potential benefits of detecting base modification,
using SMRT sequencing, include:
Single-base resolution detection of a wide
two successive base incorporations, are altered by
the presence of a modified base in the DNA
template
3
. This is observable as an increased space
between fluorescence pulses, which is called the
interpulse duration (IPD), as shown in Figure 2.
Figure 2. Principle of detecting modified DNA bases during SMRT sequencing. The presence of the modified base in the DNA
template (top), shown here for 6-mA, results in a delayed incorporation of the corresponding T nucleotide, i.e. longer
interpulse duration (IPD), compared to a control DNA template lacking the modification (bottom).
3
http://www.pacificbiosciences.com/pdf/microbial_primer.pdf

Pink Berry Genomics
PB-PSB1
(Purple sulfur bacteria)
PB-SRB1
(Sulfate reducing bacteria)
(sulfate)
(sulfide)
Wilbanks, E.G. et al (2014). Environmental Microbiology
Lizzy Wilbanks
@lizzywilbanks

99
This diagram shows a protein nanopore set in an electrically resistant membrane bilayer. An ionic current is passed through the
nanopore by setting a voltage across this membrane. If an analyte passes through the pore or near its aperture, this event creates
a characteristic disruption in current. By measuring that current it is possible to identify the molecule in question. For example, this
system can be used to distinguish the four standard DNA bases and G, A, T and C, and also modified bases. It can be used to
identify target proteins, small molecules, or to gain rich molecular information for example to distinguish the enantiomers of
ibuprofen or molecular binding dynamics.
From Oxford Nanopores Web Site
Single Molecule III: Oxford Nanopores

• Figure6. BiologicalnanoporeschemeemployedbyOxfordNanopore.
(A)SchematicofRHLproteinnanoporemutantdepictingthepositionsofthe cyclodextrin (at residue 135) and glutamines (at residue
139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the
cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave single nucleotides
from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL
protein nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand
sequencing: ssDNA is threaded through a protein nanopore and individual bases are identified, as the strand remains intact.
Panels A, B, and D reprinted with permission from ref 91. Copyright 2009 Nature Publishing Group. Panels C and E reprinted
with permission from Oxford Nanopore Technologies (Zoe McDougall).
100
nalytical Chemistry REVIEW
etected across a metal oxide-silicon layered structure. The translocated through a solid-state nanopore. In addition, sever
igure 6. Biological nanopore scheme employed by Oxford Nanopore. (A) Schematic of RHL protein nanopore mutant depicting the positions of t
yclodextrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations
he arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave sing
ucleotides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein nanopo
hat shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded through
rotein nanopore and individual bases are identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Copyrig
009 Nature Publishing Group. Panels C and E reprinted with permission from Oxford Nanopore Technologies (Zoe McDougall).
Figure6. Biological nanopores cheme employed by Oxford Nanopore.(A) Schematic of RHL protein nano pore mutant depicting the positions of the cyclodextrin (at residue
135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the cysteines. (C)
Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave single nucleotides from the target DNA strand and pass them through the
nanopore. (D) A residual current-vs-time signal trace from an RHL protein nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and
dCMP). (E) Strand sequencing: ssDNA is threaded through a protein nanopore and individual bases are identified, as the strand remains intact. Panels A, B, and D reprinted
with permission from ref 91. Copyright 2009 Nature Publishing Group. Panels C and E reprinted with permission from Oxford Nanopore Technologies (Zoe McDougall).

101
Analytical Chemistry REVIEW
would result, in theory, in detectably altered current flow through
the pore. Theoretically, nanopores could also be designed to
measure tunneling current across the pore as bases, each with a
distinct tunneling potential, could be read. The nanopore ap-
lipid bilayer, using ionic current blockage method. The autho
predicted that single nucleotides could be discriminated as lon
as: (1) each nucleotide produces a unique signal signature; (2
the nanopore possesses proper aperture geometry to accomm
Figure 5. Nanopore DNA sequencing using electronic measurements and optical readout as detection methods. (A) In electronic nanopore scheme
signal is obtained through ionic current,73
tunneling current,78
and voltage difference79
measurements. Each method must produce a characteristic sign
to differentiate the four DNA bases. Reprinted with permission from ref 83. Copyright 2008 Annual Reviews. (B) In the optical readout nanopore desig
each nucleotide is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during translocation of the DN
fragment through the nanopore. Reprinted from ref 82. Copyright 2010 American Chemical Society.
Nanopore DNA sequencing using electronic measurements and optical readout as detection methods.(A)In electronic nanopore schemes, signal is obtained
through ionic current,73 tunneling current, and voltage difference measurements. Each method must produce a characteristic signal to differentiate the four
DNA bases. (B) In the optical readout nanopore design, each nucleotide is converted to a preset oligonucleotide sequence and hybridized with labeled
markers that are detected during translocation of the DNA fragment through the nanopore.

102
Oxford Nanopores MinIon
“It’s kind of a cute device,” says Jaffe of the MinION, which is roughly the size and shape of a packet of chewing
gum. “It has pretty lights and a fan that hums pleasantly, and plugs into a USB drive.” But his technical review is
mixed. From http://www.nature.com/news/data-from-pocket-sized-genome-sequencer-unveiled-1.14724

Disclosure: Not Huge Fan of Nanopore …
103

Yet, Must Accept They Are Not a Myth
104
Pics Provided by Nick Loman

minION prep (via Nick Loman)
105
End-repair (15mins) dA-tailing (15mins)
Fragment (2mins) Clean up (5mins)
Clean up (5mins)
Ligation (10mins) Tether annealing (10mins)
HP motor incubation (30mins)
Total: 92 minutes
Slide Provided by Nick Loman

Real Time Sequencing?
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Evolution of DNA Sequencing by Jonathan Eisen

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